Abstract:

Symbiotic relationships of bacteria with higher organisms are
commonly observed in nature; however, the functional role of these
relationships is only rarely understood. This is particularly evident in
epibiotic bacterial associations in the marine environment where the bacteria
are often a diverse ensemble of microorganisms, thus complicating the
identification of the functionally important members. Classical
microbiological techniques, relying primarily on culturing these organisms,
have provided an incomplete picture of these relationships. Molecular
genetic techniques, focusing on the analyses of bacterial 16S rRNA sequences
cloned directly from natural microbial populations, are now available which
allow a more thorough examination of these associated bacterial populations.
This study sought to characterize the epibiotic bacterial population associated
with a very unique organism, Alvinella pompejana, using such a molecular
approach.
Alvinella pompejana is a polychaetous annelid that inhabits active
deep-sea hydrothermal vent sites along the East Pacific Rise. This worm
colonizes the walls of actively venting high temperature chimneys and is
thought to be one of the most thermotolerant metazoans known. The
chimney environment is characterized by high concentrations of sulfide and
heavy metals in the vicinity of the worm colonies. A morphologically
diverse epibiotic microflora is associated with the worm's dorsal integument,
with a highly integrated filamentous morphotype clearly dominating the
microbial biomass. It has been suggested that this bacterial population
participates in either the nutrition of the worm or in detoxification of the
worm's immediate environment; however, previous studies have been
unable to confirm such a role. The primary goal of this study is to
phylogenetically characterize the dominant epibionts through the analysis of
16S rRNA gene sequences.
Nucleic acids were extracted from bacteria collected from the dorsal
surface of Alvinella pompejana. 16S rRNA genes were amplified with
universal bacterial primers by the polymerase chain reaction (PCR). These
genes were subsequently cloned and the resulting clone library was screened
by restriction fragment length polymorphism (RFLP) analysis to identify
unique clone types. Thirty-two distinct clone families were found in the
library. Four of these families were clearly dominant, representing over 65%
of the library. The main assumption in this study is that the numerical
dominance of the phylotypes in the starting population will be reflected in
the clone library. Thus, representative clones from the four most abundant
clone families were chosen for complete gene sequencing and phylogenetic
analysis. These gene sequences were analyzed using a variety of phylogenetic
inference methods and were found to be related to the newly established epsilon subdivision of the Proteobacteria. In future studies, these gene
sequences will be used to construct specific oligodeoxynucleotide probes
which can be used to confirm the morphology of the clone types in the
epibiont population.